The diagnosis of drug resistant tuberculosis (TB) is hampered by financial constraints, inadequate infrastructure, the slow growth rate of Mycobacterium tuberculosis, and the genetic complexity of antibiotic resistance. Only a fraction of tuberculosis cases diagnosed globally is screened for drug susceptibility, thus hundreds of thousands of TB cases are treated with inadequate regimens of anti-TB drugs. The consequences are catastrophic and include high levels of morbidity and mortality, amplification of resistance, and increasing levels of disease transmission. The emergence of the extensively drug resistant TB, XDR-TB, has prompted an international call for better use of current diagnostics and the development of novel diagnostics. Accordingly, the WHO approved the use of Line Probe assays for the detection of M. tuberculosis complex and isoniazid and/or rifampin. These novel assays established a new diagnostic paradigm by using rapid molecular methods that identified mutations at the DNA level, rather than far slower cell culture methods. However, Line Probe assays are technically demanding and require use of sophisticated laboratories to reduce the risk of cross contamination. To circumvent this problem the WHO recently approved a contained PCR test that detects the presence of M. tuberculosis complex and rifampin resistance within 2 hours. An added advantage of this system is that it can detect M. tuberculosis complex in some smear negative cases. But, this new test only measures rifampin resistance and thus currently only serves to prompt follow-up drug susceptibility testing by conventional cell culture methods, again delaying diagnosis. The study described here is aimed at overcoming these profound barriers by transferring a pioneering technology to South Africa, where it will be evaluated using well characterized M. tuberculosis samples held in the repository at Stellenbosch University, as well as, routine clinical specimens from the National Health Laboratory service. The technology, known as LATE-PCR with Lights-On/Lights-Off Probes and PrimeSafe, invented by Professor Wangh at Brandeis University (Massachusetts), allows for the simultaneous, closed-tube detection of mutations conferring resistance to isoniazid, rifampin, ethambutol, fluorquinolones and aminoglucosides, and mycobacterial species causing disease. This technology has the potential of revolutionizing the field of molecular diagnostics for drug susceptibility in many infectious diseases by rapidly and affordably defining a profile of mutations which can be used to optimize drug treatment at initial diagnosis. Thus this technology has the potential to reduce morbidity and mortality and to help stem the global TB pandemic.